1. Field of the Invention
The present invention relates to conjugates suitable for inducing in an individual immunological tolerance to an antigen. The conjugates function by suppressing the formation of antibodies to a specific antigen. Antigens can be defined as macromolecules which will, when introduced into an individual, cause the production of antibodies by that individual and will react specifically with those antibodies.
The basic function of the organs, cells and molecules that comprise the immune system is to recognize and to eliminate from the body foreign substances. These foreign substances are eliminated by reaction between the foreign substance and antibodies which are formed in response to the substance. In general, this function is performed efficiently and without detriment to the host. However, in certain instances, disturbances can occur which can lead to pathogenic disorders such as, for example, an uncontrolled response (allergic disorders) or an abnormal response (autoimmune disease). The pathogenesis of both these disorders is related directly or indirectly to the production of antibodies to either environmental antigens (allergens) or self-antigens. In addition, since the function of the immune system is to recognize and eliminate foreign substances, transplantation of healthy tissue and organs from a donor to a genetically non-identical (i.e., allogenic) acceptor individual is difficult to achieve because of the allograft reaction.
If an individual undergoes an "altered state" as a result of contact with an antigen (and formation of antibodies thereto), subsequent contact with that antigen or a structurally similar substance can evoke a pathological reaction. Such individuals are termed "hypersensitive", with regard to one or more specific reaction-provoking antigens. When these individuals inhale or ingest the appropriate antigen, a prominent and common manifestation includes hayfever, asthma or hives. The tendency to develop this form of allergy ("atopy") is hereditable.
An individuals first antibody response to an antigen evokes a smaller and somewhat different antibody response than the response evoked upon subsequent exposure. The first exposure to an antigen evokes a primary response. After antibody levels in the primary response have declined, even to point of no longer being detectable, a subsequent encounter with the same antigen usually evokes an enhanced secondary (anamnestic) response.
The appearance of this atopy is involved with the production within an individual of a type of tissue-sensitizing IgE antibody called a reagin. These IgE antibodies have a high affinity for receptors on cells present in various body tissues. The receptors are on mast cells which are found in close association with capillaries in connective tissues throughout the body and on basophilic leukocytes (blood cells). Mast cells and basophils contain a high content of pharmacologically-active mediators, such as histamine, serotonin, (5-hydroxytryptamine) and kinins (basic peptides), concentrated in cytoplasmic granules. Contact of the IgE antibodies, which are fixed to mast cells and basophils, with antigens can trigger cross-linking of the IgE antibodies. In turn, this cross-linking causes degranulation of mast cells and basophils, which releases the chemical mediators and produces manifestations of the allergic response referred to earlier.
While the appearance of atopy is dependent upon production of cell-bound (IgE) antibodies, another type of antibodies of importance to the immune system is of the IgG class. These IgG antibodies are referred to as circulating antibodies or "blocking" antibodies. IgG antibodies are also capable of combining with antigens. This combination can inactivate antigens by "blocking" the ability of the antigen to react with cell-bound IgE, and subsequent cross-linking of the IgE antibodies.
A common method of treating allergic disorders is by immunizing ("desensitizing") the individual by repeated injections of small, increasing amounts of antigen, at intervals, e.g., weekly, and at dosage levels that avoid triggering of the degranulation of mast cells or basophils. It is believed that repeated injections increase the level of blocking IgG antibodies, but not the level of cell-bound IgE antibodies.
This desensitizing approach is subject to a number of disadvantages. Therapeutic benefits are difficult to achieve consistently, and the treatment is tedious. In addition, since exposure to the environmental antigen causes subsequent production of IgE antibodies, the possibility of an IgE-antigen reaction and subsequent IgE cross-linking, is always present.
An autoimmune disease is a pathological condition arising from an autoimmune response in which an individual responds immunologically by production of antibodies to a self-antigen. Autoimmunity can affect almost every part of the body, and generally involves a reaction between a self-antigen and IgG antibody. Representative autoimmune diseases can involve the thyroid, gastric mucosa, adrenals, skin, red cells and synovial membranes.
For some types of autoimmune diseases, non-specific immunosuppressant treatment, such as whole body X-irradiation or the administration of cytotoxic drugs, has been used with limited success. The disadvantages of such treatment include the toxicity of the agents used, and the increased incidence of various cancers, especially lymphomas and reticulum cell sarcomas, following such therapy. In addition, the use of nonspecific agents for chronic immunosuppression greatly increases the susceptibility of the patient to serious infection from environmental fungi, bacteria and viruses which under ordinary circumstances would not cause problems. The invention disclosed herein is specific and only suppresses the antibody response to the offending antigen.
In contrast to the "blocking" desensitization approach of treatment of environmental allergies with antigen extracts, and the non-specific immunosuppression of autoimmune diseases, the present invention provides a means of inducing a long-lasting state of specific immunological tolerance by suppression of formation of antibodies to specific antigens.
2. Prior Art
In viewing the prior art in the field of immunology, it is important to recognize the distinction between an antigen and a hapten. As defined hereinbefore, antigens cause the production of antibodies and also react specifically with the antibody produced. In contrast, haptens are defined as a small molecule which by itself cannot stimulate antibody production, but will combine with an antibody, once formed. Further, haptens as a rule do not induce cellular immunity, cannot serve as carriers for other haptens and induce antibody formation only when introduced on immunogenic carriers. An anti-hapten antibody response has strict carrier specificity. A secondary response to the hapten can only be elicited when it is administered on the same carrier; if it is introduced on an immunologically unrelated carrier, an individual will shown no immunological memory for the hapten and will give a typical primary response.
Because the capacity for a secondary response can persist for many years it can provide a long-lasting immunity. The primary response is less protective, because antibodies appear more slowly. In a series of papers one of the inventors and his co-workers demonstrated and characterized a system of prolonged hapten-specific bone-marrow derived cell tolerance using the D-glutamic acid:D-lysine copolymer to which the appropriate hapten had been attached. [See J. Exp. Med., Vol. 134, pp. 201-223 (1971); Vol. 136, pp. 1404-1429 and pp. 426-438 (1972); Vol. 138, pp. 312-317 (1973); Vol. 139, pp. 1446-1463 (1974) and Proc. Natl Acad. Sci. U.S.A. Vol. 71, pp. 3111-3114].
These studies demonstrated success in inducing tolerance in the bone-marrow derived lymphocyte precursors of antibody forming cells of antibody classes which were specific for the 2,4dinitrophenyl (Dnp) hapten. The investigations involved the use of conjugates of Dnp and D-glutamic acid:D-lysine, (hereinafter D-GL).
Recent studies by one of the inventors and co-workers demonstrated that tolerance to nucleoside determinants could be obtained by using conjugates of nucleosides and D-GL. [See J. Immunol., Vol. 114, pp. 872-876 (1975)]. The nucleoside work dealt with the induction of tolerance to a mixture of nucleosides, which are made up of a heterocyclic base and a five-carbon sugar. This work is similar to induction of tolerance to DNP-D-GL.
While these immunological studies are of interest in demonstrating suppression of antibody response to chemical moieties which function as single determinants when coupled to appropriate non-immunogenic carriers such as D-GL copolymer, no immunotherapeutic application to antigens is disclosed.
The experimental results herein which relate to penicillin allergy, and the use of the major antigenic determinant, benzylpenicilloyl (BPO), are described in Proc. Natl. Acad. Sci. U.S.A., Vo. 73, No. 6. pp. 2091-2095 (1976).